Laboratory of Silkworm Genetic Resources, Institute of Genetic Resources, Graduate School of Bio Resources and Bioenvironmental Science, Kyushu University, Fukuoka, 812-8581, Japan.
Laboratory of Silkworm Genetic Resources, Institute of Genetic Resources, Graduate School of Bio Resources and Bioenvironmental Science, Kyushu University, Fukuoka, 812-8581, Japan.
Insect Biochem Mol Biol. 2018 Aug;99:11-16. doi: 10.1016/j.ibmb.2018.04.008. Epub 2018 May 25.
Translucency of the larval integument in Bombyx mori is caused by a lack of uric acid in the epidermis. Hime'nichi translucent (ohi) is a unique mutation causing intermediate translucency of the larval integument and male-specific flaccid paralysis. To determine the gene associated with the ohi mutation, the ohi locus was mapped to a 400-kb region containing 29 predicted genes. Among the genes in this region, we focused on Bombyx homolog of mammalian Gephyrin (BmGphn), which regulates molybdenum cofactor (MoCo) biosynthesis, because MoCo is indispensable for the activity of xanthine dehydrogenase (XDH), a key enzyme in uric acid biosynthesis. The translucent integument of ohi larvae turned opaque after injection of bovine xanthine oxidase, which is a mammalian equivalent to XDH, indicating that XDH activity is defective in ohi larvae. RT-PCR and sequencing analysis showed that (i) in ohi larvae, expression of the BmGphn gene was repressed in the fat body where uric acid is synthesized, and (ii) there was no amino acid substitution in the ohi mutant allele. Finally, we obtained BmGphn knockout alleles (hereafter denoted as BmGphn) by using CRISPR/Cas9. The resulting ohi/BmGphn larvae had translucent integuments, demonstrating that BmGphn is the gene responsible for the ohi phenotype. Our results show that repressed expression of BmGphn is a causative factor for the defective MoCo biosynthesis and XDH activity observed in ohi larvae. Interestingly, all male BmGphn homozygotes died before pupation and showed a flaccid paralysis phenotype. The genetic and physiological mechanisms underlying this flaccid paralysis phenotype are also discussed.
家蚕幼虫表皮缺乏尿酸导致体壁半透明。Hime'nichi translucent（ohi）是一种独特的突变，导致幼虫体壁半透明，且雄性特有的瘫痪。为了确定与 ohi 突变相关的基因，将 ohi 基因座定位到一个包含 29 个预测基因的 400kb 区域。在这个区域的基因中，我们重点关注哺乳动物 Gephyrin 的家蚕同源物（BmGphn），因为 Gephyrin 调节钼辅因子（MoCo）生物合成，而 MoCo 是黄嘌呤脱氢酶（XDH）的活性所必需的，XDH 是尿酸生物合成的关键酶。ohi 幼虫的半透明体壁在注射牛黄嘌呤氧化酶后变得不透明，黄嘌呤氧化酶是 XDH 的哺乳动物等价物，这表明 ohi 幼虫的 XDH 活性有缺陷。RT-PCR 和测序分析表明：（i）在 ohi 幼虫中，BmGphn 基因在合成尿酸的脂肪体中表达受到抑制；（ii）ohi 突变等位基因没有氨基酸取代。最后，我们通过使用 CRISPR/Cas9 获得了 BmGphn 敲除等位基因（此后表示为 BmGphn）。产生的 ohi/BmGphn 幼虫体壁半透明，证明 BmGphn 是 ohi 表型的相关基因。我们的结果表明，BmGphn 的表达受到抑制是 ohi 幼虫中观察到的 MoCo 生物合成和 XDH 活性缺陷的一个原因。有趣的是，所有雄性 BmGphn 纯合子在化蛹前死亡，并表现出瘫痪的表型。还讨论了这种瘫痪表型的遗传和生理机制。
